Isotopically labeled alpha-keto acids and esters

ABSTRACT

Isotopically labeled alpha-keto acids and esters are disclosed herein. Also disclosed are methods of synthesizing isotopically labeled alpha-keto acids and esters.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 60/830,301 filed Jul. 11, 2006 andU.S. Provisional Patent Application No. 60/851,706 filed on Oct. 13,2006, the disclosures of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

Pyruvic acid (C₃H₄O₃) has the chemical name 2-oxopropanoic acid, and hasa molecular mass of 88.06 grams per mol.

Pyruvic acid is a colorless organic liquid formed as an intermediate incarbohydrate metabolism and as an end product in glycolysis. Pyruvicacid has a melting point ranging from about 11° C. to about 12° C. andis soluble in water.

In the laboratory, pyruvic acid may be prepared by heating a mixture oftartaric acid and potassium hydrogen sulfate, or by the hydrolysis ofacetyl cyanide, formed by a reaction of acetyl chloride with potassiumcyanide. Production under these conditions, however, leaves undesirableimpurities, which can be toxic or harmful if not removed in entirety.

Pyruvic acid also occurs naturally as an intermediate product incarbohydrate and protein metabolism in the human body. Pyruvic acid isimportant in metabolism as it can be converted to carbohydrates viagluconeogenesis, to fatty acids or energy through acetyl-CoA (which isthe main input for a series of reactions known as the Krebs cycle), tothe amino acid alanine, and to ethanol.

In industry, pyruvic acid is used to produce its salts and esters(pyruvates) for the use as dietary supplements and as an effective meansof weight loss. Pyruvic acid is also used for the synthesis of aminoacids and used for biomedical research. Its derivatives are used inmaking food additives and flavoring agents.

Unfortunately, due to the highly reactive nature of pyruvic acid,storage of the molecule over extended periods of time is very difficultand undesirable.

U.S. Pat. No. 6,753,446 describes diethyl oxalate analogs useful forasymmetric labeling of synthetic compounds. The compounds have thegeneral structure RO—C(O)C(O)—X

United States Patent Publication No. 2007/0106085 describesintermediates useful in the preparation of [¹³C₁₋₅]metacrylic acid.

United States Patent Publication No. 2006/0178534 describes labeledcompounds useful for the preparation of labeled compounds, includingpyruvic acid.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention there isprovided an isotopically labeled compound of Formula (IIa),

wherein:

X is F, Cl, Br, I, MgF, MgCl, MgBr and MgI;

each R and each R¹ may be the same or different and independently mayrepresent hydrogen, deuterium, tritium or a C₁-C₃₆ substituted orunsubstituted, saturated, or unsaturated, linear, branched, cyclic,aromatic, or substituted aromatic group, wherein R or R¹ may include aheteroatom including O, N, S, Si, and P wherein any of the carbon atomsor heteroatoms may be isotopically labeled; and

y is independently 12, 13, or 14.

In accordance with another embodiment of this invention, each R and eachR¹ are independently selected from the group consisting of hydrogen,deuterium, and a C₁-C₄ straight chain or branched alkyl.

Recently, isotopically enriched pyruvic acid was studied for its use ina variety of medical diagnostic applications. The use of pyruvic acidthat is isotopically enriched with at least one carbon 13 isotope allowsfor its use in medical diagnostics. Due to the highly reactive nature ofpyruvic acid, storage of the molecule over extended periods of time isdifficult and undesirable. Fortunately, storage of pyruvic acid ispossible by forming shelf stable precursors of pyruvic acid, and thenconverting the precursor to pyruvic acid prior to its use, for example,as a medical diagnostic agent.

Disclosed are two synthetic pathways for the synthesis of precursors ofpyruvic acid. The synthesis described herein provides pyruvic acidprecursors of the present invention and derivatives in high yield whichare shelf stable and which are of a high purity. Moreover, the pyruvicacid precursors allow conversion to pyruvic acid in one step. Finally,the synthetic methodologies of the present invention avoid the use ofdangerous reagents, such as potassium cyanide.

DETAILED DESCRIPTION

The present invention provides compounds having Formula (I)

wherein:G represents a halogen or a Grignard-halogen complex such as MgF, MgBr,MgCl, and MgI;

wherein ⁺A represents a positively charged counterion;

Q represents C or O, each of which may be isotopically labeled;

Q′ represents O or N, each of which may be isotopically labeled;

each R, each R¹, and each R² may be the same or different andindependently may represent hydrogen, deuterium, tritium or a C₁-C₃₆substituted or unsubstituted, saturated, or unsaturated, linear,branched, cyclic, aromatic, or substituted aromatic group, wherein R orR¹ may include a heteroatom including O, N, S, Si, and P wherein any ofthe carbon atoms or heteroatoms may be isotopically labeled;

m is 1 if Q′ is O or 2 if Q′ is N;

n is 0 if Q is O or 2 if Q is C; and

y is independently 12, 13, or 14;

wherein the compound is not unlabeled pyruvic acid, the salts of pyruvicacid, unlabeled benzyl pyruvate, unlabeled benzyl methacrolate,propanoic-3-¹³C acid-2-oxo-phenylmethyl ester; or 2-propenoicacid-2-(methyl-¹³C-d3)-phenylmethyl ester.

As used herein, the terms “isotope”, “isotopic” or “isotopicallylabeled” refer to an atom having the same number of protons but adifferent number of neutrons as compared with the most abundant form ofthe element. Accordingly, carbon may be isotopically labeled as ¹³C,nitrogen may be isotopically labeled as ¹⁵N, sulfur may be isotopicallylabeled as ³²S and oxygen may be isotopically labeled as ¹⁶O, ¹⁷O or¹⁸O. These terms as used herein also refer to radio-labeled elements.Further, these terms as used herein also refer to molecules whichcontain isotopic atoms.

The terms “aromatic” or “cyclic group” as used herein, encompasses notonly the group but also the substitutions in one or more positions.Substitutions may include, and without limitation, halogens, hydroxyl,nitro, amino, substituted amino having the formula —N(R³)(R³), (whereinR³ is a C₁-C₆ linear, branched, or cyclic alkyl group), C₁-C₅ alkoxy, orC₁-C₅ alkyl groups. Thus, for example, a reference to a benzyl group caninclude, for example, meta-chloro benzene, 3,4,5 tri-bromo benzene,p,m,o-methyl, p,m,o-methoxy, and trifluoromethyl.

Hydrogen atoms, which by convention are not shown, may be deuterium ortritium.

In a preferred embodiment, at least one atom in Formula (I) is isotopic.More preferably, at least one carbon or ^(y)C is C¹³ or C¹⁴.

The present invention also provides compounds of Formula (II)

wherein:

X represents F, Cl, Br, I, MgF, MgCl, MgBr and MgI;

Q represents C which may be isotopically labeled;

each R, each R¹, and each R² may be the same or different andindependently may represent hydrogen, deuterium, tritium or a C₁-C₃₆substituted or unsubstituted, saturated, or unsaturated, linear,branched, cyclic, aromatic, or substituted aromatic group, wherein R orR¹ may include a heteroatom including O, N, S, Si, and P wherein any ofthe carbon atoms or heteroatoms may be isotopically labeled;

n is 2; and

y is independently 12, 13, or 14.

In some embodiments, each R and each R¹ each may be the same ordifferent and independently may be selected from hydrogen, deuterium,tritium or a C₁-C₂₄ substituted or unsubstituted, saturated orunsaturated, linear, branched, cyclic, aromatic or substituted aromaticmoiety optionally containing one or more heteroatoms including O, N, S,P, and Si, any of which may be isotopically labeled.

In other embodiments, each R and each R¹ may be the same or differentand independently may be selected from hydrogen, deuterium, tritium or aC₁-C₁₆ substituted or unsubstituted, saturated or unsaturated, linear,branched, cyclic, aromatic or substituted aromatic moiety optionallycontaining one or more heteroatoms including O, N, S, P, and Si, any ofwhich may be isotopically labeled.

In yet other embodiments, each R and each R¹ may be the same ordifferent and independently may be selected from hydrogen, deuterium,tritium or a C₁ to C₆ linear or branched, substituted or unsubstituted,cyclic, or aromatic moiety, optionally containing one or moreheteroatoms including O, N, S, P, and Si, any of which may beisotopically labeled.

In preferred embodiments of the compositions of Formula (II), each R,each R¹, and each R² may be the same or different and independently maybe selected from hydrogen, deuterium, methyl, ethyl, propyl, isopropyl,butyl, secbutyl, tertbuytl, allyl, 2-butenyl, 3-butenyl, phenyl, benzyl,napthyl, cyclopropyl, cyclopentyl, and cyclohexyl, thienyl, furyl,pyridyl, imidazoylyl, benzimidazoyl, or benzothiazolyl.

In a preferred embodiment, at least one atom in Formula (II) isisotopic. More preferably, at least one carbon is ¹³C or ¹⁴C. Mostpreferably, at least one ^(y)C is ¹³C or ¹⁴C.

In some embodiments, the compounds of Formula (II) have the structure ofFormula (IIa):

wherein R and R¹ are as defined previously.

In other embodiments, the compounds of Formula (II) have the structureof Formula (IIb):

wherein R and R¹ are as defined previously.

Non-limiting examples of compounds falling within the scope of thecompounds of Formula (II) include:

The present invention also provides compounds of Formula (III)

wherein:

Q represents C or O, each of which may be isotopically labeled;

Q′ represents O, or N, each of which may be isotopically labeled;

each R, each R¹, and each R² may be the same or different andindependently may represent hydrogen, deuterium, tritium or a C₁-C₃₆substituted or unsubstituted, saturated, or unsaturated, linear,branched, cyclic, aromatic, or substituted aromatic group, wherein R orR¹ may include a heteroatom including O, N, and S, wherein any of thecarbon atoms or heteroatoms may be isotopically labeled;

m is 1 or 2;

n is 0 or 2; and

y is independently 12, 13, or 14;

wherein the compound is not unlabeled pyruvic acid, the salts of pyruvicacid, unlabeled benzyl pyruvate, unlabeled benzyl methacrolate,propanoic-3-¹³C acid-2-oxo-phenylmethyl ester; or 2-propenoicacid-2-(methyl-¹³C-d3)-phenylmethyl ester.

In a preferred embodiment, at least one atom in Formula (III) isisotopic. More preferably, at least one carbon or ^(y)C is C¹³ or C¹⁴

In some embodiments, each R, each R¹, and each R² may be the same ordifferent and independently may be selected from hydrogen, deuterium,tritium or a C₁-C₂₄ substituted or unsubstituted, saturated orunsaturated, linear, branched, cyclic, aromatic or substituted aromaticmoiety optionally containing one or more heteroatoms including O, N, S,Si, and P any of which may be isotopically labeled.

In other embodiments, each R, each R¹, and each R² may be the same ordifferent and independently may be selected from hydrogen, deuterium,tritium or a C₁-C₁₆ substituted or unsubstituted, saturated orunsaturated, linear, branched, cyclic, aromatic or substituted aromaticmoiety optionally containing one or more heteroatoms including O, N, andS, any of which may be isotopically labeled.

In yet other embodiments, each R, each R¹, and each R² may be the sameor different and independently may be selected from hydrogen, deuterium,methyl, ethyl, propyl, isopropyl, butyl, secbutyl, tertbuytl, allyl,2-butenyl, 3-butenyl, phenyl, benzyl, napthyl, cyclopropyl, cyclopentyl,cyclohexyl, thienyl, furyl, pyridyl, imidazoylyl, benzimidazoyl, orbenzothiazolyl.

In yet further embodiments, R² is selected from a protecting groupincluding but not limited to methoxymethyl ether, tetrahydropyranylether, t-Butyl ether, allyl ether, benzyl ether, trimethylsilyl ethers,triethylsilyl ethers, t-butyldimethylsilyl ether, t-butyldiphenylsilylether, acetic acid ester, benzoic acid ester, methylthiomethyl ethers,benzyloxymethyl ethers, 2-napthylmethyl ethers, p-methoxybenzyl ethers,trityl ethers, and methoxytrityl ethers.

In some embodiments, Q is C; Q′ is O; and R² is H, and at least one^(y)C group is ¹³C or ¹⁴C.

In other embodiments, Q is C; Q′ is O; and R² is benzyl, and at leastone ^(y)C group is ¹³C or ¹⁴C.

In further embodiments, Q is O; Q′ is O; and R² is benzyl, and at leastone ^(y)C group is ¹³C or ¹⁴C.

In yet other embodiments, the compounds of Formula (III) have thestructure of Formula (IIIa):

wherein ^(y)C, R, R¹, and R² are as defined previously.

In yet further embodiments, the compounds of Formula (III) have thestructure of Formula (IIIb):

wherein ^(y)C, R, R¹, and R² are as defined previously.

In preferred embodiments, the compounds of Formula (III) have thestructure of Formula (IIIc)

wherein:

Q, ^(y)C, R, and R¹ are as defined previously; and

R⁴ represents a C₁-C₁₀ aromatic ring optionally substituted with one ormore nitro, amino, substituted amino having the formula —N(R³)(R³),halogen, deuterium, tritium, C₁-C₄ alkoxy, or C₁-C₄ alkyl groups,wherein the aromatic ring optionally includes a heteroatom selected fromthe group consisting of O, N, and S.

Examples of aromatic rings representative of R⁴ include, but are notlimited to, phenyl, napthyl, benzofuran, isobenzofuran, indole,benzothiophenee, benzimidazole, indazole, benzoxazole, benzisoxazole,benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline,pyridazine, cinnoline and the substituted variants thereof.

It has been found that the inclusion of a single carbon spacer adjacentto R⁴, as in Formula (IIIc) and the Formulas which follow, allows forcleavage of the R² group (Formula (III)) by means of hydrogenationrather than acid hydrolysis. As discussed herein, hydrogenation providesan efficient means by which the compounds of Formula (IIIc) caneventually be converted to pyruvic acid or its derivatives thereof.Moreover, hydrogenation allows for the production of pyruvic acid underneutral conditions and where the only byproduct is toluene. Othermethods, including acid hydrolysis, leave inorganic or organic acids asimpurities.

In more preferred embodiments, the compounds of Formula (III) have thestructure of Formula (IIId):

wherein Q, ^(y)C, R, and R¹ each are as defined previously; and R⁵represents at most 5 substitutions on the aromatic ring, wherein R⁵independently represents nitro, amino, substituted amino having theformula —N(R³)(R³), halogen, deuterium, tritium, C₁-C₄ alkoxy, or C₁-C₄alkyl group.

In one particularly preferred embodiment, the compounds of Formula(IIId) have the structure of Formula (IIIe):

wherein ^(y)C, R, R¹, and R⁵ are as defined previously.

In another particularly preferred embodiment, the compounds of Formula(IIId) have the structure of Formula (IIIf):

wherein ^(y)C, R¹, and R⁵ are as defined previously.

In another particularly preferred embodiment, the compounds of Formula(IIIe) have the structure of Formula (IIIg):

wherein ^(y)C, R, and R⁵ are as defined previously.

In another particularly preferred embodiment, the compounds of Formula(IIIe) have the structure of Formula (IIIh):

wherein ^(y)C and R⁵ are as defined previously.

In yet another particularly preferred embodiment, the compounds ofFormula (IIIf) have the structure of Formula (IIIi):

wherein ^(y)C and R⁵ are as defined previously.

In a most preferred embodiment of Formula (IIIi), each R⁵ is hydrogen.

In yet another particularly preferred embodiment, the compounds ofFormula (IIId) have the structure of Formula (IIIj):

wherein ^(y)C, Q, n, R, R¹, and R⁵ are as defined previously.

As illustrated below in Formula (IIIj), in a preferred embodiment inaccordance with the present invention and particularly for thoseisotopically labeled compounds discussed herein having the structuresgenerally shown in Formulas IIIa through IIIi, as well as Formula V, itis preferred that the double bond oxygen on the ester forming carbonyl,the carbon of the ester forming carbonyl and/or the carbon of themethacrylate or ketone group be isotopically labeled. In a particularlypreferred embodiment in accordance with the present invention, theisotopic labeling of the compounds described above would occur at someatom other than the carbon bound to the various R¹ groups. Isotopicallylabeling may occur at a plurality of other groups as well.

Non-limiting examples of the compounds of Formulas (IIIe) and Formula(IIIf) include:

In other embodiments, the compounds of Formula (I) have the structure ofFormula (IV):

wherein ^(y)C, Q, R, and R¹ are as defined previously.

Non-limiting examples of the compounds of Formulas (IV) include:

wherein R represents R² of Formula (IV).

In the propamides of Formula (IV) above, R is preferably a C₁-C₄ alkyl,more preferably methyl or ethyl.

The present invention also provides a method of synthesizingisotopically labeled compounds including analogs of pyruvic acid.

One synthetic method, according to the following scheme, comprisesreacting a compound of Formula (IIa) with magnesium turnings in asolvent to yield a compound having Formula (IIb):

Preferably, the solvent is an aprotic solvent. More preferably, thesolvent is an ether or toluene.

Preferably, the reaction is run at room temperature, more preferably thereaction is initially run at room temperature with a subsequent increasein temperature to drive the reaction to completion. As used herein,“room temperature” means a temperature ranging from about 22° C. toabout 26° C.

In a preferred embodiment, each R and each R¹ may be the same ordifferent and independently may be selected from hydrogen or C₁-C₄alkyl, more preferably each R and each R¹ are hydrogen.

Another synthetic method, according to the following scheme, comprisesreacting a compound of Formula (IIb)) with labeled or unlabeled carbondioxide in a solvent to yield a compound having Formula (IIIa′):

Preferably, the solvent is selected from ether, tetrahydrofuran,dioxane, and glymes.

Preferably, the reaction is run with cooling, more preferably at atemperature of about 0° C. or below, most preferably at about −50° C. orbelow.

In a preferred embodiment, each R and each R¹ may be the same ordifferent and independently may be selected from hydrogen and C₁-C₄alkyl, more preferably each R and each R¹ are hydrogen.

Another synthetic method, according to the following scheme, comprisesreacting a compound of Formula (IIIa′) with a weak base in the presenceof a reagent having a halogenated leaving group to yield a compoundhaving Formula (IIIb):

Preferably, bases include K₂CO₃, NaHCO₃, Li₂CO₃, Cs₂CO₃, t-ButOK,t-ButOLi, hydroxides, alkyl salts, lithium salts, metal hydrides, andheteroatom bases. More preferably the base is K₂CO₃.

In a preferred embodiment, R²X is benzyl chloride or benzyl bromide.

In a preferred embodiment, the reaction is run at room temperature orbelow. One skilled in the art would recognize that the reaction may berun at temperatures lower than room temperature (between roomtemperature and −78° C.) to accommodate certain bases.

In a preferred embodiment, each R and each R¹ may be the same ordifferent and independently may be selected from hydrogen and C₁-C₄alkyl, more preferably each R and each R⁴ are hydrogen.

In a preferred embodiment R² is —CH₂—R⁴, wherein R⁴ includes, but is notlimited, to phenyl, napthyl, benzofuran, isobenzofuran, indole,benzothiophenee, benzimidazole, indazole, benzoxazole, benzisoxazole,benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline,pyridazine, cinnoline and the substituted variants thereof.

Yet another synthetic method, according to the following scheme,comprises reacting a compound for Formula (IIIa) with ozone in a solventto yield a compound having Formula (IIIb):

Preferably, this reaction is run in a C₁-C₆ alcohol (straight chain orbranched), methylene chloride, chloroform and the like.

Preferably, the reaction is run at a reduced temperature, morepreferably at about 0° C. or below. As used herein, the term “reducedtemperature” refers to a temperature below room temperature.

In a preferred embodiment, each R and each R¹ may be the same ordifferent and independently may be selected from hydrogen and C₁-C₄alkyl, more preferably each R and each R¹ are hydrogen.

In a preferred embodiment R² is —CH₂—R⁴, wherein R⁴ includes but is notlimited to, phenyl, napthyl, benzofuran, isobenzofuran, indole,benzothiophenee, benzimidazole, indazole, benzoxazole, benzisoxazole,benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline,pyridazine, cinnoline and the substituted variants thereof. In a morepreferred embodiment, R² is benzyl.

Yet a further synthetic method, according to the following scheme,comprises converting a compound having Formula (IIIb) to isotopicallylabeled pyruvic acid or an analog thereof by means of hydrogenation,preferably in a solvent selected from a C₁-C₆ alcohol (straight chain orbranched), a ketone, water, an ether, or mixtures thereof.

The reaction is preferably run under a pressure of about 4 to about 12psig, more preferably the reaction is run using a palladium catalyst,even more preferably the palladium catalyst is on charcoal.

The reaction is preferably run at room temperature.

In a preferred embodiment, each R¹ is independently selected fromhydrogen or C₁-C₄ alkyl, more preferably each R¹ is hydrogen.

The above conversion can also be carried out via acid hydrolysis usingtechniques known to those of skill in the art. United States PatentPublication No. 2006/0178534, incorporated herein by reference,describes a method of performing acid hydrolysis. For example, acidhydrolysis may be accomplished by treating a compound with 1M HCl andthen extracting the final product with an organic solvent, preferablyethyl acetate.

Yet a further synthetic method, according to the following scheme,comprises converting a compound having Formula (IIIf) to isotopicallylabeled pyruvic acid or an analog thereof by means of hydrogenation,preferably in a solvent selected from a C₁-C₆ alcohol (straight chain orbranched), a ketone, water, an ether, or mixtures thereof.

The reaction is preferably run under a pressure of about 4 to about 12psig, more preferably run using a palladium catalyst, even morepreferably the palladium is on charcoal.

In a preferred embodiment, each R¹ is independently selected fromhydrogen or C₁-C₄ alkyl, more preferably each R¹ is hydrogen.

Yet a further synthetic method, according to the following scheme,comprises converting a pyruvate salt or an analog thereof to therespective pyruvate analog of Formula (IIIb) as follows:

Preferably, the solvent is a polar aprotic solvent. More preferably, thesolvent is dimethylformamide.

In a preferred embodiment, each R¹ is independently selected fromhydrogen or C₁-C₄ alkyl, more preferably each R¹ is hydrogen.

Preferably R²X is R²Br. More preferably, R²X is benzyl bromide.

This method of converting an isotopically labeled pyruvate salt to therespective pyruvate analog is particularly useful in isolating orpurifying pyruvate salts from materials which contain dimerized pyruvicacid.

The compounds of Formula (IIIb), formed occurring to the reaction, areseparated by techniques known in the art, preferably by distillation orchromatography.

The compounds of Formula (IIIa) synthesized by this process can then beconverted to the respective isotopically labeled pyruvic acid analogs asdescribed previously or even converted to the same pyruvate salt (buthaving a higher purity than the original pyruvate salt).

Accordingly, one synthetic route to pyruvic acid or the intermediates ofpyruvic acid using the compounds generally described in Formulas (I),(II), and (III) is as follows:

In general, magnesium turnings are combined with Formula IIa to yieldthe Gringard reagent Formula IIb. Formula IIb is then reacted in thepresence of isotopically labeled carbon dioxide (wherein the oxygenatoms may optionally be isotopically labeled) to yield the intermediateof Formula IIIa′. This reaction can be run in an organic solventselected from ether and related solvents including tetrahydrofuran,dioxane, glymes and the like.

The reaction is preferably run with cooling and more preferably at atemperature of about 0° C. or below, more preferably about −50° C. orbelow. The reaction is also preferably run in an inert atmosphere.

Intermediate Formula IIIa′ is then reacted with a weak base in a solventincluding methylene chloride, THF, or acetone at room temperature orabove and with a reagent having a halogenated leaving group to arrive atthe compound of Formula IIIa. One of ordinary skill in the art would beable to determine the necessary reagent having a halogenated leaving toarrive at the desired pyruvate derivative. For example, the reagenthaving a halogenated leaving group may be selected from benzyl chloride.Bases useful in the processes of the present invention include K₂CO₃,NaHCO₃, Li₂CO₃, Cs₂CO₃, t-ButOK, t-ButOLi, hydroxides, metal hydrides,and alkyl salts.

Ozonolysis is then carried out in the presence of Formula IIIIa to yieldthe compound of Formula IIIb. Preferably, this reaction can be run in aC₁-C₆ alcohol (straight chain or branched), methylene chloride,chloroform and the like at a reduced temperature, more preferably atabout 0° C. or below.

Finally, the compound of Formula IIIb is hydrogenated to yield theisotopically labeled pyruvic acid of Formula V. This can be run in aC₁-C₆ alcohol (straight chain or branched), a ketone, water, an ether,or mixtures thereof. The reaction is preferably run under a pressure ofabout 4 to about 12 psig, more preferably run using a palladiumcatalyst, even more preferably on charcoal. The hydrogenation could alsobe run in acetone.

This step could also be undertaken using by means of acid hydrolysisunder conditions known to those of skill in the art.

Although the scheme shown above is one method of producing pyruvic acid,one of skill in the art would understand that any of the intermediatesdescribed above can be synthesized, isolated, and recoveredindependently of one another. The intermediates may be used to producepyruvic acid intermediates or other compounds not disclosed herein.

Isotopically labeled pyruvic acid analogs can also be prepared byhydrolyzing compounds having Formula (IV) under acidic conditions. Bymeans of example, the production of [1-¹³C]pyruvic acid by means of acidhydrolysis is illustrated below.

Compounds having Formula (IV) can be produced from isotopically labeledN,N-dialkyl-2-oxo-oxamates by Grignard addition, as follows:

Synthesis of [1-¹³C]Propanamide, N,N-dialkyl-2-Oxo

wherein R is as defined as R² in Formula (IV).

Synthesis of [2-¹³C]Propanamide, N,N-dialkyl-2-Oxo

wherein R is as defined as R² in Formula (IV).

Synthesis of [1,2-¹³C₂]Propanamide, N,N-dialkyl-2-Oxo

wherein R is as defined as R² in Formula (IV).

Starting materials for the above conversion to compounds having Formula(IV) may be produced in accordance with U.S. Pat. No. 6,753,446,incorporated herein by reference. For example, an oxamide may beprepared according to the following: [¹³C]Methyl phenyl sulfide wasreacted with sec-butyl lithium followed by [¹³C]carbon dioxide to formintermediate (I). This intermediate (I) was then reacted with oxalylchloride followed by dimethyl amine to form intermediate (II). Thisintermediate (II) was then reacted with sulfuryl chloride followed by 10percent water in ethanol to form [¹-¹³C]acetic acid,(dimethylamino)oxo-, ethyl ester.

Compounds of Formula (IV) can also be prepared by reacting compoundshaving Formula (IIIa′) with oxalyl chloride to form an acid halideintermediate, followed by reaction with an amine to form thedi-substituted amide having Formula (IV). For example, the reaction withoxalyl chloride, and amine, proceeds as follows:

Preferably, the reaction with oxalyl chloride is run in a non-polaraprotic solvent, more preferably dichloromethane. Preferably, thereaction with the amine is run in a non-polar solvent, more preferablyTHF.

Synthetic examples of the production of isotopically labeled benzylpyruvate and isotopically labeled pyruvic acid are as follows:

Step 1: Synthesis of [1-¹³C]methacrylic acid

An oven dried 2 L 3-neck round bottom flask equipped with a 300 mmAllihn reflux condenser with gas adapter, heating mantle, 125 mLaddition funnel with septa, and mechanical stirrer was placed undervacuum and back filled with Argon. Isopropenyl magnesium bromide (0.6moles) was then added to the round-bottom flask, and cooled andmaintained at—keep with 78 C with a dry ice/acetone bath. The ¹³CO₂ wasbubbled into the Grignard via a needle and measured with a flow meterset at about 200 mL per minute, 27 g, 0.6 moles. The addition took about55 minutes. After addition, the reaction was stirred for fifteenminutes. Meanwhile, 100 mL of concentrated 12M HCl, 1.2 moles, wasdiluted with 100 mL of water and transferred to the addition funnel inportions and added as a steady stream to the Grignard over about tenminutes. After addition, the cold bath was removed and replaced withwater, to warm the reaction to room temperature. The resulting mostlycolorless biphasic mixture was transferred to a 2 L separatory funnel.The lower aqueous phase was made acidic by addition of 0.6 moles of HCl.The aqueous was extracted with dichloromethane (3×200 mL) and wasseparated. The organic layers were combined and washed with 0.7 moles ofNaOH. The aqueous layer was separated and evaporated to dryness. The[1-¹³C]sodium methacylate was used in the subsequent reaction withoutpurification.

Step 2: Synthesis of Benzyl [1-¹³C]methacrolate

The [1-¹³C]sodium methacylate was added to dimethyl formamide (DMF) (250mL) in a 2 L round bottom flask at room temperature. This mixture wasallowed to stir for about five minutes and then benzyl chloride (77.195g, 0.6098 mol, 70.18 mL) was added at a quick drip rate over a twelveminute period. The reaction proceeded for six hours. Dichloromethane(750 mL) was added to the mixture and this mixture was filtered througha frit funnel. It was then transferred to a separatory funnel and waswashed with DI water (3×150 mL). The last wash (4th) was done usingsodium thiosulfate (15 g in 150 mL) to remove iodine from the solution.The organic extract was dried over sodium sulfate and evaporated invacuo to a slightly yellow oil containing the desired product with someDMF. The DMF was removed under vacuum by heating the flask to 40oC untilabout all of the DMF solvent was removed.

Step 3: Synthesis of Benzyl [1-13C]pyruvate

A stirred solution of 25 mL (147.5 mmole) of benzyl methacrylate in 500mL of dichloromethane and 125 mL of methanol was cooled to −78° C. andozonized until the solution was pale blue, indicating excess ozone. Thesolution was purged with nitrogen until the blue color of ozone haddissipated, and then 14.1 mL (192 mmole, 1.3 equivalents) of dimethylsulfide was added rapidly dropwise under nitrogen. After stirring forone hour more at −78° C. the solution was removed from the cold bath andallowed to stir at room temperature for 3 hours. The solution may bestored overnight in the freezer at this point. Volatiles were removed onthe rotary evaporator at 40° C. and the residue was taken up in 100 mLof dichloromethane and washed with 100 mL of water to remove dimethylsulfoxide. The water layer was back extracted with a small volume ofdichloromethane. The combined organics were washed with water in thisfashion twice more. The final organic layer was filtered through cotton,concentrated on the rotary evaporator, and high-vacuum dried leaving anessentially quantitative yield of benzyl pyruvate as a colorless liquid.A small amount of formaldehyde methyl hemiacetal and/or the methylhemiacetal of benzyl pyruvate may be present but do not interfere in thenext step.

Step 4: Synthesis of [1-13C]Pyruvic Acid

5 g (28 mmole) of the above benzyl pyruvate was dissolved in 100 mL ofabsolute ethanol in a heavy-walled bottle and blanketed with nitrogen.0.5 g of 51 palladium on charcoal catalyst was added and the mixture wasin a Parr shaker hydrogenation apparatus. The mixture was deaeratedthree times by evacuation followed by refilling with hydrogen.Hydrogenation was then commenced at 8 psi for one hour. Hydrogen wasthen removed by evacuation followed by refilling with nitrogen. Catalystwas removed by vacuum filtration through a bed of Celite. The filtercake was washed with ethanol and the colorless to pale yellow filtratewas concentrated on the rotary evaporator at room temperature. Excessivevacuum or higher temperature must be avoided to prevent loss of product.The product obtained still contained a little ethanol and showed pyruvicacid, varying amounts of its ethyl hemiacetal, and a very small amountof ethyl pyruvate by NMR. Water may be added and distilled off at a bathtemperature of less than 50° C. under high vacuum with liquid nitrogencooling of the receiver. After most of the water was removed, additionalwater was added to the pot and the process repeated. What remained was aconcentrated solution of pyruvic acid and its hydrate possiblycontaminated by a little formaldehyde hydrate and ethyl pyruvate.Titration with 1N aqueous sodium hydroxide to an endpoint of pH 5.8 andremoval of water and other volatiles yielded solid sodium pyruvate.

The reaction above could also be run in other solvents for example thereaction was run using acetone and at the end of the process[1-13C]Pyruvic acid was isolated as a mixture of 2 parts pyruvic acid to1 of acetone. For example, the Hydrogenation of benzyl [1-¹³C]pyruvateusing acetone is accomplished as follows: Benzyl [1-¹³C]pyruvate (5.1 g,0.0285 moles) was dissolved in acetone (51 mL) and placed in ahydrogenation vessel which had 10% Pd/carbon (0.28 g). The reaction waspurged with argon and then the reaction was evacuated under vacuum. Thereaction vessel was filled with hydrogen to 10 psi and then shaken for24 hours. The reaction was filtered to remove the catalyst and thenconcentrated. This mixture, which now contained toluene and[1-¹³C]pyruvic acid, was treated with hexane. The layers were thenseparated and the hexane layer which contained the [1-¹³C]pyruvic acidwas evaporated to give a quantitative yield of the desired product as amixture of acetone to [1-¹³C]pyruvic acid (1:2).

Shelf Stability of Isotopically Labeled Pyruvic Acid:

Samples of isotopically labeled [1-¹³C]pyruvic acid, having Formula VIand synthesized as discussed immediately below, were subjected tovarious conditions as depicted in Table 1, in order to establish thecompound's shelf storage stability.

The [1-¹³C]pyruvate of Formula VI was synthesized as follows:

Benzyl [1-¹³C]pyruvate, Formula VIA, was synthesized according to themethods discussed previously. The benzyl [1-¹³C]pyruvate washydrogenated and the hydrogenation mixture was diluted with water andthen extracted with dichloromethane. The organic phase contained tolueneand some methanol while the aqueous phase contained the [1-¹³C]pyruvicacid and methanol. The aqueous phase was then distilled until all of themethanol was removed, thereby leaving [1-¹³C]pyruvic acid (purity:98+/−2%, by NMR).

The resulting [1-¹³C]pyruvic acid was tested as follows in Table 1:

TABLE 1 Samples of isotopically labeled pyruvic acid were subjected tovarious storage conditions. Condition Duration Sample TemperatureStorage 12 weeks 17 weeks 1 −10° C. Dark Stable Stable 2  4° C. DarkStable Stable 3 Room Dark Stable Stable temperature* 4 Room Light StableStable temperature* *Room temperature denotes a temperature ranging fromabout 22° C. to about 26° C.

¹H and ¹³C NMR spectra of the distilled [1-¹³C]pyruvic acid in water(0.025M) were acquired at the onset of the study and prior to exposingthe samples to the various conditions specified in Table 1. ¹H and ¹³CNMR spectra were again acquired for each of the samples after beingexposed to the aforementioned conditions on a weekly basis for 12-weeks.The spectra acquired after exposure were compared visually and appearedto remain unchanged as compared with the spectra acquired prior toexposure, i.e. the spectra were as expected for [1-¹³C]pyruvic acid inwater. As such, it was concluded that each of the samples were shelfstable. Similarly, ¹H and ¹³C NMR spectra acquired after 17-weeks ofexposure remained unchanged as compared with the spectra acquired priorto exposure. Once again, it was concluded that each of the samples wereshelf stable under the conditions provided in Table 1.

Concentration studies were also performed to show that [1-¹³C]pyruvicacid solutions would also be expected to be stable at higherconcentrations. To demonstrate such stability, the [1-¹³C]pyruvic acidsolutions were compared to commercial samples of pyruvic acid (FormulaVIB) obtained from Sigma-Aldrich Co. in water at higher concentrations.Commercial pyruvic acid was dissolved in water to arrive at the variousconcentrations listed in Table 2 below. Each of these commercialsolutions were subjected to light for 6-weeks at temperatures rangingfrom about 22° C. to about 26° C. At each of the concentrations listedbelow, it was discovered that the pyruvic acid solutions were stable. Asthe purity of the commercial pyruvic acid samples were comparable to thepurity of the materials produced in accordance with the presentinvention, and as the commercial materials were stable in water for atleast 6 weeks at relatively high concentrations, it is believed that[1-¹³C]pyruvic acid solutions having at least those concentrations inwater would also be stable.

TABLE 2 Pyruvic acid, of varying concentrations, was found to be stableafter 6-weeks. (Formula VIB)

Concentration After 6-weeks 11.3 Stable 5.7 Stable 3.8 Stable 2.8 Stable2.3 Stable

The molarity of sample 4 was confirmed to be 0.025M by titrating theaqueous sample with sodium hydroxide. The titration with the sodiumhydroxide also revealed that in acidic conditions, such as at pHsranging from about 5.8 to about 1, pyruvic acid may be present either asthe acid or as sodium [1-¹³C]pyruvate, depending on the sodium hydroxideconcentration. It was also discovered that at pHs greater than 5.8, suchas at a pH of about 6.2, some of the pyruvic acid was present in theform of a dimerized sodium salt. For example, at a pH of about 6.2, thepH attained in titrating the solution of sample 4, HPLC revealed thatsample 4 consisted of 89% sodium [1-¹³C]pyruvate and about 2-4%dimerized [1-¹³C]pyruvate salt (accounting for about 4-6% of the pyruvicacid). As such, it was discovered that to minimize dimmer formation, itmay be necessary to control the pH of [1-¹³C]pyruvate solutions, and inparticular, any aqueous solution should be maintained at a pH of about6.5 or less, more preferably 6.0 or less, and even more preferably 5.8or less.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

1. An isotopically labeled compound of Formula (IIa),

wherein: X is F, Cl, Br, I, MgF, MgCl, MgBr and MgI; each R and each R¹may be the same or different and independently may represent hydrogen,deuterium, tritium or a C₁-C₃₆ substituted or unsubstituted, saturated,or unsaturated, linear, branched, cyclic, aromatic, or substitutedaromatic group, wherein R or R¹ may include a heteroatom including O, N,S, Si, and P wherein any of the carbon atoms or heteroatoms may beisotopically labeled; and y is independently 12, 13, or
 14. 2. Theisotopically labeled compound of claim 1, wherein at least one of said^(y)C is selected from the group consisting of ¹³C or ¹⁴C.
 3. Theisotopically labeled compound of claim 1, wherein all of said ^(y)C are¹²C.
 4. The isotopically labeled compound of claim 1, wherein each R andeach R¹ are independently selected from the group consisting ofhydrogen, deuterium, and a C₁-C₄ straight chain or branched alkyl. 5.The isotopically labeled compound of claim 4, wherein each R and each R¹are hydrogen.
 6. The isotopically labeled compound of claim 5, whereinat least one of said ^(y)C is selected from the group consisting of ¹³Cor ¹⁴C.
 7. The isotopically labeled compound of claim 1, wherein X isMgX.
 8. The isotopically labeled compound of claim 7, wherein each R andeach R¹ are independently selected from the group consisting ofhydrogen, deuterium, and a C₁-C₄ straight chain or branched alkyl. 9.The isotopically labeled compound of claim 8, wherein each R and each R¹are hydrogen.
 10. The isotopically labeled compound of claim 9, whereinat least one of said ^(y)C is selected from the group consisting of ¹³Cor ¹⁴C.